Hepatitis-A viruses adapted to human fibroblast cells

ABSTRACT

Hepatitis-A virus, suitable for use in human or animal vaccines, is prepared by adapting the virus first to human kidney cells and subsequently adapting the thus-altered virus to human fibroblast strains. In the process the growth rate of Hepatitis-A virus on the noted tissues is materially increased. This HAV is also a useful antigen in diagnostic test procedures.

German patent application (DE-OS) No. P 30 33 406.6 (published Apr. 15,1982) describes a method for making Hepatitis-A Virus (HAV). The growthof HAV can now be accelerated by seeding HAV on Rhesus monkey kidneycells (FrhR-4/R), harvesting resulting viruses which appear afterseveral weeks and subsequently similarly seeding the resulting virusesagain. This process is repeated until the obtained viruses have asatisfactory rate of growth.

Resulting HAV are unsuited for vaccines for human medicine because theycome from animal cells.

SUMMARY OF THE INVENTION

There are a number of distinct aspects of this invention, all of whichmake possible vaccine (derived from Hepatitis-A virus) for humanmedicine, which is one aspect and a principal object. A furtherprincipal object is the use of the cell culture produced HAV as anantigen in diagnostic test procedures. Other aspects include:

(a) selecting tissue for sequential culturing of HAV,

(b) adapting HAV to human kidney cells (HKC),

(c) increasing the growth rate of HAV on HKC,

(d) HAV/HKC,

(e) adapting HAV/HKC to human fibroblast strains (HFS),

(f) increasing the growth rate of HAV/HKC on HFS,

(g) HAV/HFS and

(h) HAV/HFS antigens and antibodies produced therefrom.

Beyond these noted aspects, operating equipment, parameters andprocedures, tissue preparation and culturing, culture media, virusinoculation and culturing, detection and analysis methods and equipment,preparing antigens, producing antibodies, harvesting viruses andantibodies and formulating and administering vaccine are substantiallyconventional and well within the skill of the art.

The main purpose of this invention is to prepare HAV which can be usedin human medicine. Such viruses are Hepatitis-A viruses, HAV/HFS, whichare adapted to human fibroblast strains (HFS) via human embryo kidneycells (HKC). Their production is accomplished by culturing HAV on HKC,harvesting resulting viruses, culturing thus-harvested viruses on HKCand repeating this procedure to obtain HAV/HKC having an acceptablegrowth rate. The HAV/HKC is then cultured on HFS, harvesting resultingviruses and similarly repeating the culturing and harvesting to obtainHAV/HFS.

DETAILS

Attempts to propagate unmodified HAV isolate of human origin fromclinical specimens in fibroblast cultures were unsuccessful. Over aperiod of fifteen weeks no HAV was detected (by RIA orimmunofluorescence methods) in infested cells. HAV adapted to HKC overten passages (HAV/HKC), however, was successfully propagated in humanfibroblast; four weeks after inoculation with HAV/HKC the firstsupernatant of HFS (positive for HAV) was detected with RIA(radioimmunoassay). Further adaptation of HAV/HKC to human fibroblaststrains was achieved by seeding the first positive (for HAV) supernatanton HFS and repeating the procedure as was done to adapt HAV to HKC.

HAV isolated from stool (unmodified HAV) is cultured in HKC.Conventional methods, such as those described in the previously-notedpatent application No. P 30 33 406.6, are employed. The first virusesreleased (after about seven or eight weeks) are used, once again, toinoculate HKC (passage 2 in HKC). This process is repeated for about tenpassages, with an increase in growth rate resulting from each successivepassage. The viruses, which grow rapidly and in large quantities, areselected in each case.

HAV adapted to HKC (HAV/HKC) is thus obtained for the first time. TheHAV/HKC is a modified HAV.

It is most surprising that:

1. viruses can be harvested at all after a comparatively long intervalof time; as the usual rate of growth is considerably higher, theimprobability of such a process is apparent rather soon; and

2. adoption to HKC with a high rate of growth is achieved throughrepeated passages.

These procedures are repeated in connection with infection of humanfibroblast cells (HFS) with HAV/HKC. Here again, HAV/HKC grows at a verylow rate in its first passage in HFS; eventual success is thus entirelyunexpected.

By means of repeated passages of HAV/HKC on HFS, HAV is finally adaptedto HFS; in other words, HAV/HFS is eventually harvested both from thecell and from the nutrient medium.

The HAV/HFS is equally suitable for use in tests with HAV antibodiesand, above all, for making vaccine for human medicine.

The invention is explained in greater detail in the following examples:

EXAMPLE I Hepatitis-A Virus Isolates

Stool suspensions containing HAV strains, GBG, GBM and GJA, are used.The origin of these isolates is described by Flehmig et al. (1977).Stool suspensions are prepared as 2.5 percent suspensions with Hanks'balanced salt solution, as described by Flehmig et al. (1977).

The 2.5 percent suspensions are buffered at pH 7.2 with phosphatebuffered saline (PBS) and partially cleared by centrifugation at 3000 Gand again at 6000 G for 30 minutes each. The final supernatant isdesignated "fecal extract".

Cells and HAV/GBM were deposited at Paul-Ehrlich Institute,Frankfurt/Main, on Jul. 15, 1981.

EXAMPLE II Cell Cultures

Kidneys and lungs from human embryos (10 to 25 weeks old) are processedaccording to established procedures for producing human kidney cells(HKC) and human fibroblast strains (HFS). Cell cultures are establishedby conventional procedures using TC 199 Hanks' plus 10 percent fetalcalf serum for HKC and MEM Hanks' plus 7 percent fetal calf serum forHFS as growth media. It is possible to subpass the first monolayerstratum of kidney cells at least 5 times, e.g. from 5 to 10 times, andof fibroblast strains over 50 times, e.g. from 50 to 75 times. Parts ofthe first passage of HKC and of HFS are optionally frozen according towell-known methods at -70° C. or in liquid nitrogen, thawed when neededand subsequently used.

EXAMPLE III Cultivating HAV in HKC and HFS

Densely-grown monolayer strata of HKC or HFS are incubated withHAV-containing suspensions, and the viruses are absorbed by the cells.After that, suitable nutrient medium is placed on cells, which areincubated at from 34° to 37° C. After corresponding incubation times,the quantity of HAV, which is extracted from the cells into the nutrientmedium, is determined by means of suitable conventional analysismethods. The extracted HAV is useful for test methods to establish thepresence of antibodies in human or animal serums or, according tocertain purification and inactivation methods, for vaccine for man andanimals.

EXAMPLE IV Adaptation of HAV to HKC

Five weeks after inoculating HKC (cf. Example II), with HAV (firstpassage through), the first supernatant (positive for HAV) is detectedby RIA. To select the more rapidly growing viruses, the firstHAV-positive supernatant detected for each passage is used as inoculumfor the next passage on HKC. The time between inoculation of HAV anddetection of the first HAV-positive supernatant is gradually reducedwith each successive passage.

The first HAV-positive supernatant obtained (first passage) for HKCinoculated with HAV is detected, e.g., by RIA 34 days after infection ofthe cells. In the tenth passage the first HAV-positive supernatant isdetected by RIA about 6 days after infection.

Cell-bound viruses are harvested by freezing and thawing the cells threetimes with 10 percent (by volume) of the supernatant to produce aconcentrate. The amount of HAV in the resulting thawed product is thusgreater than that previously in cell-bound form.

In this experiment the first cell lysate positive for HAV (ninth passagethrough HKC) was detected by RIA five days after infection; the firstsupernatant positive for HAV, eight days after infection. In anotherexperiment the first supernatant positive for HAV (ninth passage throughHKC) was detected six days after infection; the first supernatantpositive for HAV (18th passage through HKC) was detected by RIA fourdays after inoculation.

HAV adapted to HKC during ten passages is designated HAV/HKC.

EXAMPLE V Adaptation of HAV to HFS

Following the procedures of Example IV, HFS is inoculated with HAV/HKC,the first supernatant positive to HAV/HKC is used to inoculate HFS, andthe steps are repeated until Hepatitis-A viruses adapted to humanfibroblast cells (HAV/HFS) are obtained.

To produce HAV/HFS for vaccine purposes an effective amount ofimmunity-inducing HAV-antigen is used.

EXAMPLE VI Harvesting Cell-Bound Virus and Detecting Virus in theSupernatant

Remove maintenance medium from cells and cultured virus in a 75 ccflask. Add 3 ml of Hanks' balanced salt solution to the flask. Freezeand thaw (three times) the cells and Hanks' solution. Clear theresulting product by centrifugation for 30 minutes at 3,000×G. Use theobtained supernatant for detecting HAV/AG by RIA. The virus antigendetected this way is regarded as cell-bound virus.

To measure the amount of virus in the supernatant, 100 microliters ofmaintenance medium is subject to RIA without prior treatment.

EXAMPLE VII Radioimmunoassay (RIA) for HAV Detection

Using the RIA procedure of Purcell et al. (1976); which has beendescribed in detail by Flehmig (1978) and by Flehmig et al. (1978), asolid phase (PVC U-plates, Dynatech, Nuertingen/FRG) is coated with anANTI-HAV. After three wash steps, the suspension to be tested for HAV ispoured into the wells. After an overnight incubation period at roomtemperature, the cups are washed and a purified human IgG preparationwith a high concentration of ANTI-HAV labeled with radioactive I-125(150,000 counts per minute (cpm)) is added into two wells together withan ANTI-HAV positive serum and into two wells together with an ANTI-HAVnegative serum. The microliter plates are subsequently incubated at 37°C. for 6 hours.

Antigen samples are considered to be positive if values greater than 2.1are obtained by dividing the CPM of two test samples from ANTI-HAVnegative serum by the CPM of two test samples from ANTI-HAV positiveserum; the P/N ratio by the competitive binding technique with ANTI-HAVpositive and ANTI-HAV negative serum insures the specificity of eachtest run.

EXAMPLE VIII Radioimmunoassay for Detection of ANTI-HAV

For measuring ANTI-HAV IgG, the solid-phase is coated with ANTI-HAV inthe manner referred to in Example VII. A defined amount of HAV is boundto the antibody layer in the previously-described manner. 0.05 mi ofserum to be tested and 0.05 ml of I-125-labeled ANTI-HAV IgG are placedinto each cup simultaneously.

Reduction in residual radioactivity, as compared with a pre-illnessserum, is determined. A reduction of 50 percent or more is considered tobe positive.

EXAMPLE IX RIA for Detection of IgM Antibody to HAV

The solid phase (PVC-U plates) are coated with 100 microliters of rabbitantiserum to human IgM [diluted to a concentration of 5×0.01 inphosphate-buffered saline (PBS), pH 7.2, containing 0.1 percent sodium]per well. After incubation for 24 hours at room temperature (about 24°C.), 10 microliters of calf serum are added to each well to saturate thesolid phase. The coated plates are then used directly or stored forseveral weeks at 4° C. Sera to be tested for IgM antibody to HAV areserially diluted: 0.01, 0.001, 0.0001, 0.00001 and 0.000001, in PBS, pH7.2. After the wells are rinsed three times with PBS (pH 7.2), each wellis charged with 100 microliters of each dilution of serum. Nonspecificbinding is determined with eight wells by adding 100 microliters of 1percent pooled human serum devoid of antibody to HAV diluted in 3percent calf serum in PBS.

After incubation for from 6 to 8 hours at 37° C., the wells are washedthree times with PBS, pH 7.2. Then each well is charged with 100microliters of a suspension of HAV, and the plates are incubatedovernight at room temperature. After three more washes, 100 microlitersof a solution of (I-125) IgG antibody to HAV (giving 150,000 CPM) isadded to each well, and the plates are incubated at 37° C. for 6 hours.Finally, the wells are washed three times with PBS (pH 7.2), and theradioactivity of each well is measured with a gamma counter [Kontron,Munich, German Federal Republic (FRG)]. Sample counts exceeding fivetimes the counts obtained with the nonspecific-binding wells areconsidered to be significant (positive for antibody to HAV). Afteroptimization, the preceding procedure was performed.

The same principles prevail when the employed tracer is an ANTI-HAV-IgGor Anti-HAV-F(ab)2 labelled with peroxidase or phosphatase.

Hepatitis-A viruses (HAV) adapted to human) fibroblast strains(designated HAV/HFS) are deposited with the Paul Ehrlich Institute,Frankfurt/Main.

The invention and its advantages are readily understood from thepreceding description. Various changes may be made in the procedures,equipment, operating parameters and testing procedures without departingfrom the spirit and scope of the invention or sacrificing its materialadvantages. The processes and products hereinbefore described are merelyillustrative of preferred embodiments of the invention.

What is claimed is:
 1. A process which comprises culturing unmodifiedHAV on human kidney cells (HKC) and harvesting .[.the thus-.]. produced.Iadd.cell-free .Iaddend.viruses.
 2. A process which comprises culturingon HKC isolated .Iadd.cell-free .Iaddend.viruses produced by the processof claim 1 and isolating thus-produced .Iadd.cell-free .Iaddend.viruses.3. A process for increasing the growth rate of HAV cultured on HKC whichcomprises (a) the process of claim 2, (b) culturing the resultingisolated .Iadd.cell-free .Iaddend.viruses on HKC and isolatingthus-produced .Iadd.cell-free .Iaddend.viruses and (c) repeating step(b) a number of times, each with isolated .Iadd.cell-free.Iaddend.viruses from the immediately preceding culturing on HKC.
 4. Aprocess of claim 3 which comprises from about five to ten successiveculturing steps.
 5. A process according to claim 2 or claim 3 whichcomprises isolating from the final culturing step those viruses whichgrow most rapidly and/or are produced in largest quantities.
 6. Aprocess according to claim 2 or claim 3 which comprises (a) selectingfrom each culturing step those viruses which grow most rapidly and/orare produced in largest quantities and (b) culturing only thus-selectedviruses in any immediately succeeding culturing step.
 7. A process ofclaim 1 wherein the HAV is Hepatitis-A virus isolate of human origin. 8.A process of claim 7 wherein the HAV consists essentially of thatisolated from stool.
 9. A process of claim 1 which consists essentiallyof culturing HAV on HKC.
 10. Modified HAV adapted to HKC (HAV/HKC) andhaving a rate of growth and/or a rate of proliferation significantlygreater than that of unmodified HAV cultured on HKC.
 11. A process whichcomprises culturing the modified virus of claim 10 on human fibroblaststrains (HFS).
 12. A process which comprises culturing on HFS isolatedviruses produced by the process of claim 11 and isolating thus-producedviruses.
 13. A process for increasing the growth rate of HAV/HKCcultured on HFS which comprises (a) the process of claim 12, (b)culturing the resulting isolated viruses on HFS and isolatingthus-produced viruses and (c) repeating step (b) a number of times, eachwith isolated viruses from the immediately preceding culturing on HFS.14. A process of claim 13 which comprises from about 50 to 75 successiveculturing steps.
 15. A process according to claim 13 or claim 14 whichcomprises (a) selecting from each culturing step those viruses whichgrow most rapidly and/or proliferate in greatest quantities and (b)culturing only thus-selected viruses in any immediately succeedingculturing step.
 16. Modified HAV according to claim 10 and furtheradapted to HFS (HAV/HFS) and having a rate of growth and/or a rate ofproliferation significantly greater than that of modified HAV accordingto claim .[.8.]. .Iadd.10 .Iaddend.and cultured on HFS.
 17. A processwhich comprises:(a) culturing unmodified HAV on human kidney cells (HKC)and harvesting thus-produced viruses, (b) culturing isolated virusesfrom step (a) on EIKC and isolating thus-produced viruses, (c) culturingisolated viruses from step (b) on HKC and isolating thus-producedviruses, (d) repeating step (c) a number of times, each with isolatedviruses from the immediately preceding culturing on HKC to obtain HAVadapted to HKC (HAV/HKC) and having a rate of growth and/or a rate ofproliferation significantly greater than that of HAV cultured on HKC,the isolated viruses cultured in and isolated from the products of steps(b) through (d) being those which grow most rapidly and/or are producedin largest quantities; (e) culturing HAV/HKC isolated from step (d) onhuman fibroblast strains (HFS) and isolating thus-produced viruses, (f)culturing isolated viruses from step (e) on HFS and isolatingthus-produced viruses, (g) culturing isolated viruses from step (f) onHFS and isolating thus-produced viruses, (h) repeating step (g) a numberof times, each with isolated viruses from the immediately precedingculturing on HFS to obtain HAV adapted to HFS (HAV/HFS) and having arate of growth and/or a rate of proliferation significantly greater thanthat of HAV/HKC cultured on HFS, the isolated viruses cultured in andisolated from the products of steps (e) through (h) being those whichgrow most rapidly and/or are produced in largest quantities. .Iadd. 18.A process which comprises culturing unmodified HAV on human kidney cells(HKC) in a culture medium until supernatant in the culture medium ispositive for HAV, inocculating fresh HKC with the supernatant, culturingagain until supernatant is positive for HAV, and harvestingthus-produced viruses from the latter supernatant. .Iaddend. .Iadd. 19.A process of claim 1 which comprises culturing unmodified HAV on humankidney cells (HKC) and harvesting, directly from culture supernatant,produced cell-free viruses which are free from cell particles withoutfurther separation. .Iaddend. .Iadd.20. A process of claim 1 whichcomprises culturing unmodified HAV on human kidney cells (HKC) untilcell-free viruses pass through intact cell walls into culturesupernatant, and harvesting produced cell-free viruses from thesupernatant. .Iaddend.